Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing [version 2; peer review: 2 approved]

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work

Predictive models for mutations in mismatch repair genes: implication for genetic counseling in developing countries

<p>Abstract</p> <p>Background</p> <p>Lynch syndrome (LS) is the most common form of inherited predisposition to colorectal cancer (CRC), accounting for 2-5% of all CRC. LS is an autosomal dominant disease characterized by mutations in the mismatch repair genes mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), postmeiotic segregation increased 1 (PMS1), post-meiotic segregation increased 2 (PMS2) and mutS homolog 6 (MSH6). Mutation risk prediction models can be incorporated into clinical practice, facilitating the decision-making process and identifying individuals for molecular investigation. This is extremely important in countries with limited economic resources. This study aims to evaluate sensitivity and specificity of five predictive models for germline mutations in repair genes in a sample of individuals with suspected Lynch syndrome.</p> <p>Methods</p> <p>Blood samples from 88 patients were analyzed through sequencing MLH1, MSH2 and MSH6 genes. The probability of detecting a mutation was calculated using the PREMM, Barnetson, MMRpro, Wijnen and Myriad models. To evaluate the sensitivity and specificity of the models, receiver operating characteristic curves were constructed.</p> <p>Results</p> <p>Of the 88 patients included in this analysis, 31 mutations were identified: 16 were found in the MSH2 gene, 15 in the MLH1 gene and no pathogenic mutations were identified in the MSH6 gene. It was observed that the AUC for the PREMM (0.846), Barnetson (0.850), MMRpro (0.821) and Wijnen (0.807) models did not present significant statistical difference. The Myriad model presented lower AUC (0.704) than the four other models evaluated. Considering thresholds of ≥ 5%, the models sensitivity varied between 1 (Myriad) and 0.87 (Wijnen) and specificity ranged from 0 (Myriad) to 0.38 (Barnetson).</p> <p>Conclusions</p> <p>The Barnetson, PREMM, MMRpro and Wijnen models present similar AUC. The AUC of the Myriad model is statistically inferior to the four other models.</p

Experience of taking care of children exposed to HIV: a trajectory of expectations

OBJETIVO: conhecer a experiência do cuidador/mãe em relação ao cuidado para com a criança exposta ao vírus da imunodeficiência humana por transmissão vertical, na trajetória pós-natal. MÉTODO: pesquisa qualitativa, que utilizou o Interacionismo Simbólico como referencial teórico. Foram realizadas entrevistas com 39 cuidadores de crianças expostas ao vírus da imunodeficiência humana e em seguimento em um serviço especializado. Os dados foram analisados pela análise de conteúdo indutiva. RESULTADOS: apreenderam-se quatro categorias que reportam à experiência solitária de manuseio da terapia antirretroviral da criança, no que se refere principalmente à ausência ou incompletude de informações recebidas; estar atento aos cuidados, que incluem o uso da profilaxia para pneumonia, vacinas e outros restritos à interação mãe/criança; querer omitir a presença do vírus da imunodeficiência humana pelo medo do preconceito e olhar o futuro e temer a doença. CONCLUSÃO: a presença do vírus da imunodeficiência humana e a ameaça dessa infecção na criança são capazes de gerar apreensão e tantos outros sentimentos como medo, culpa e ansiedade no cuidador. Os profissionais de saúde precisam trabalhar conjuntamente com a mãe para o enfrentamento das demandas e sofrimentos. Assim, o tratamento para evitar a transmissão vertical será eficiente e a mãe, juntamente à criança, viverá essa trajetória com apoio, apesar da apreensão pelo resultado.OBJETIVO: conocer la experiencia del cuidador/madre en relación al cuidado de niño expuesto al VIH por transmisión vertical en la trayectoria posnatal. MÉTODO: investigación cualitativa, que utilizó el Interaccionismo Simbólico como referencial teórico. Fueron realizadas entrevistas con 39 cuidadores de niños expuestos al VIH y que eran acompañados en un servicio especializado. Los datos fueron analizados por el análisis de contenido inductivo. RESULTADOS: se encontraron cuatro categorías que informan sobre la experiencia solitaria de la administración de la terapia antirretroviral del niño, en lo que se refiere principalmente a: la ausencia o a informaciones incompletas recibidas; al estar atento a los cuidados, que incluyen el uso de la profilaxis para neumonía, vacunas y otros restringidos a la interacción madre-niño; al querer omitir la presencia del VIH por miedo al prejuicio; y al mirar al futuro y temer a la enfermedad. CONCLUSIÓN: la presencia del VIH y la amenaza de infectar al niño son capaces de generar preocupación y varios otros sentimientos en el cuidador, como miedo, culpa y ansiedad. Los profesionales de la salud precisan trabajar conjuntamente con la madre para el enfrentamiento de las demandas y sufrimientos. Así, el tratamiento para evitar la transmisión vertical será eficiente y la madre con el niño vivirán esa trayectoria con apoyo, a pesar de la preocupación por el resultado.'OBJECTIVE: to learn about the experience of caregivers/mothers providing care to infants exposed to HIV through vertical transmission. METHODS: this qualitative study used Symbolic Interactionism as the theoretical framework. A total of 39 caregivers of children exposed to HIV in follow-up at a specialized service were interviewed. Data were analyzed through inductive content analysis. RESULTS: four categories were identified that report on the lonely experience of handling the child's antiretroviral therapy, mainly due to a lack of information or incomplete information; being attentive to required care, such as the use of prophylaxis for pneumonia, vaccines, and other practices restricted to the mother-child interaction; the desire to omit the HIV out of fear of prejudice and fear of the disease, considering future prospects. CONCLUSION: the HIV and the threat this infection may affect the child cause apprehension and feelings such as fear, guilt and anxiety in the caregivers. Healthcare workers need to work together with mothers so they are able to cope with demands and distress. Only then will the treatment to avoid vertical transmission be efficient and will mother and child be supported during the process, despite apprehension with the outcome

Static cut-points of hypertension and increased arterial stiffness in children and adolescents: The International Childhood Vascular Function Evaluation Consortium

Pediatric elevated blood pressure (BP) and hypertension are usually defined using traditional BP tables at the 90th and 95th percentiles, respectively, based on sex, age, and height, which are cumbersome to use in clinical practice. The authors aimed to assess the performance of the static cut-points (120/80 mm Hg and 130/80 mm Hg for defining elevated BP and hypertension for adolescents, respectively; and 110/70 mm Hg and 120/80 mm Hg for children, respectively) in predicting increased arterial stiffness. Using data from five population-based cross-sectional studies conducted in Brazil, China, Korea, and New Zealand, a total of 2546 children and adolescents aged 6-17 years were included. Increased arterial stiffness was defined as pulse wave velocity >= sex-specific, age-specific, and study population-specific 90th percentile. Compared to youth with normal BP, those with hypertension defined using the 2017 American Academy of Pediatrics guideline (hereafter referred to as "percentile-based cut-points") and the static cut-points were at similar risk of increased arterial stiffness, with odds ratios and 95% confidence intervals of 2.35 (1.74-3.17) and 3.07 (2.20-4.28), respectively. Area under the receiver operating characteristic curve and net reclassification improvement methods confirmed the similar performance of static cut-points and percentile-based cut-points (P for difference > .05). In conclusion, the static cut-points performed similarly well when compared with the percentile-based cut-points in predicting childhood increased arterial stiffness. Use of static cut-points to define hypertension in childhood might simplify identification of children with abnormal BP in clinical practice

Recent improvements in the development of A2B adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A1, A2A, A2B and A3 (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N6-, C2-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA1Ki = 1050 nM, hA2AKi = 1550 nM, hA2B EC50 = 82 nM, hA3Ki > 5 μM) and its 2-chloro analogue 23 (hA1Ki = 3500 nM, hA2AKi = 4950 nM, hA2B EC50 = 210 nM, hA3Ki > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA1, hA2A, hA3 EC50 > 10 μM; hA2B EC50 = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis

Determinants of high residual post-PCV13 pneumococcal vaccine-type carriage in Blantyre, Malawi:a modelling study

Background In November 2011, Malawi introduced the 13-valent pneumococcal conjugate vaccine (PCV13) into the routine infant schedule. Four to 7 years after introduction (2015–2018), rolling prospective nasopharyngeal carriage surveys were performed in the city of Blantyre. Carriage of Streptococcus pneumoniae vaccine serotypes (VT) remained higher than reported in high-income countries, and impact was asymmetric across age groups. Methods A dynamic transmission model was fit to survey data using a Bayesian Markov-chain Monte Carlo approach, to obtain insights into the determinants of post-PCV13 age-specific VT carriage. Results Accumulation of naturally acquired immunity with age and age-specific transmission potential were both key to reproducing the observed data. VT carriage reduction peaked sequentially over time, earlier in younger and later in older age groups. Estimated vaccine efficacy (protection against carriage) was 66.87% (95% CI 50.49–82.26%), similar to previous estimates. Ten-year projected vaccine impact (VT carriage reduction) among 0–9 years old was lower than observed in other settings, at 76.23% (CI 95% 68.02–81.96%), with sensitivity analyses demonstrating this to be mainly driven by a high local force of infection. Conclusions There are both vaccine-related and host-related determinants of post-PCV13 pneumococcal VT transmission in Blantyre with vaccine impact determined by an age-specific, local force of infection. These findings are likely to be generalisable to other Sub-Saharan African countries in which PCV impact on carriage (and therefore herd protection) has been lower than desired, and have implications for the interpretation of post-PCV carriage studies and future vaccination programs.</p